About 2% of the world population (123 million individuals) are chronically infected with the hepatitis C virus (HCV). Chronic infection puts these individuals at risk for the development of hepatitis, cirrhosis, liver failure and hepatocellular carcinoma making chronic hepatitic C the leading cause for liver transplantation worldwide. In the United States a seroprevalence rate of 1.8% has been reported and HCV is associated with more than half of an increasing number of newly diagnosed hepatocellular carcinomas.
Current therapy combining pegylated interferon-alpha with ribavirin achieves cure rates of just above 50% (Fried et al., 2002, N Engl J Med 347:975-82; Manns et al., 2001, Lancet 358:958-965). Several difficult to treat patient groups show decreased response rates or cannot tolerate therapy at all. These include patients that have failed to respond to standard therapy, African Americans, patients with HIV-coinfection or end-stage liver disease and patients after liver transplantation. Currently, HCV infection of the graft after liver transplantation is universal, usually leading to rapid fibrosis progression and subsequent graft failure. This accounts for the poor outcome of liver transplantation for HCV-induced cirrhosis compared to other indications (Forman et al., 2002, Gastroenterology 122:889-96). Targeting HCV cell entry in this setting holds promise as a therapy capable of blocking viral entry even for a short period of time might prevent graft re-infection and thus turn liver transplantation from a palliative into a curative procedure. Hopefully, a more complete understanding of early HCV life cycle events will identify promising targets for this purpose.
Recent technical developments have opened up exciting new possibilities for molecular studies of hepatitis C virus (HCV). In the past decade, molecular clones that are functional for chimpanzee infection, efficient cell culture systems for studying RNA replication (replicons), and retroviral pseudotypes harboring functional HCV glycoproteins (HCVpp) have been developed. More recently, derivatives of a genotype 2a isolate, JFH-1, have yielded relatively high titers of cell culture infectious particles (HCVcc). Importantly, HCVcc is infectious in chimpanzees and a murine-human xenograft model and viral production in animals (chHCVcc or muHCVcc) retaining infectivity in cultured cells has been shown. This creates a complete and valuable set of reagents to study HCV neutralization and entry.
The tetraspanin CD81 and scavenger receptor BI/II (SR-BI/II) are cell surface molecules that bind the HCV E2 glycoprotein and participate in HCV entry. However, expression of these two molecules, in conjunction with numerous other candidate entry factors, is insufficient to render cells fully permissive for HCV entry. Thus, there necessarily remains an additional, as yet unidentified HCV coreceptor.
HCV is a member of the family Flaviviridae, which also includes Pestiviruses and Flaviviruses. The HCV virion consists of an enveloped nucleocapsid containing the viral genome, a single, positive stranded RNA of approximately 9,600 nucleotides. Viral entry into the host cell is thought to require a tightly regulated interaction between the viral envelope proteins, E1 and E2, and host proteins at the cell surface. Moreover, it has been shown that host cell infection requires endosomal acidification suggesting that fusion of the viral envelope with cellular membranes is a pH triggered event. After cell entry the nucleocapsid is released into the cytosol and the viral RNA is translated through action of an internal ribosome entry site (IRES) present in the 5′ untranslated region (5′UTR). The HCV genome encodes a single long open reading frame giving rise to a viral polyprotein of over 3000 amino acids that then undergoes co- and post-translational proteolytic processing to generate the mature viral proteins: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (FIG. 1). The viral structural proteins, including core, the capsid protein E1, and E2, are encoded by the first third of the polyprotein. p7 (a putative ion channel) and nonstructural (NS) proteins, encoded by the C-terminal two-thirds of the polyprotein, are components of the HCV RNA replication complex. The replication of the viral genome through a negative strand RNA intermediate occurs.
Recently, pseudotyped retroviral particles were developed to study HCV entry. To generate HCVpp, 293T cells are transfected with expression vectors encoding (1) unmodified HCV E1E2, (2) the gag-pol proteins of either MLV or HIV and (3) a packaging competent (but gag-pol and env deficient) retroviral genome containing either a GFP or lacZ reporter gene. This results in the release of infectious HIV or MLV nucleocapsids surrounded by an envelope containing HCV glycoproteins (Bartosch, B., J. Dubuisson, and F. L. Cosset. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633-642). Alternatively, a two vector system using an envelope deficient HIV genome with a luciferase reporter and an HCV-E1E2 expressing vector can be employed (Hsu, M., J. Zhang, M. Flint, C. Logvinoff, C. Cheng-Mayer, C. M. Rice, and J. A. McKeating. 2003. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl. Acad. Sci. USA 100:7271-76). These HCVpp systems take advantage of the ability of retroviruses to incorporate heterologous glycoproteins into their envelope. HCVpp can infect a number of liver derived human cell lines and their reporter genes allow convenient quantification of target cell infection, making HCVpp the first robust assay for the study of HCV glycoprotein mediated cell entry.
More has been elucidated in the life cycle of HCV via the development of HCVcc (FIG. 2). This was made possible through the discovery of a genotype 2a HCV isolate from a Japanese patient with acute fulminant hepatitis (JFH)-1, that is capable of efficient subgenomic RNA replication in multiple cell types without the need for adaptive mutations (Date, T. et al. 2004. J Biol. Chem. 279:22371-6; Kato, T. et al. 2003 Gastroenterology 125:1808-17; Kato, T. et al. 2005J. Virol 79:592-6). Surprisingly, when full length JFH-1 genome RNA was transfected into Huh-7 cells, viral particles, termed HCVcc, were released that were capable of infecting naïve cells. Efficient in vitro systems based on the JFH genome recapitulating the entire HCV life cycle have been developed. Virus produced from full length JFH-1 RNA exhibits characteristics predicted for an HCV virion: the infectivity of these particles is blocked by antibodies against E2 or CD81 and by purified soluble CD81; moreover, virion density is similar to that found in sera of infected individuals. Although JFH-1 initially yielded low titers, higher viral titers were obtained by using Huh-7.5 cells and derived sublines (Lindenbach, B. D, et al. 2005 Science 309:623-6; Zhong, J. P., et al. 2005 Proc Natl Acad Sci USA 102:9294-9), which are highly permissive for HCV replication (Blight et al. 2002. J. Virol. 76:13001-14) due to a defect in the RIG-I intrinsic immune response pathway (Sumpter et al. 2005. J Virol 79:2689-99). Virion production was further enhanced through the use of a chimeric genotype 2a full length genome, expressing the core through NS2 region of the HCJ6 HCV isolate cloned into the JFH-1 genome (J6/JFH) (FIG. 1B), which, unexpectedly, produced higher initial titers post transfection than the full length JFH-1 genome (Lindenbach, B. D., et al. 2005 Science 309:623-6). Very recently, cell culture grown HCVcc has been used to infect both chimpanzees and uPA-SCID mice transplanted with human hepatocytes (Lindenbach, B. D., et al. 2006 Proc. Natl. Acad. Sci. USA 103 In press). In both cases rising viral loads and an infection sustained for several weeks ensued, validating the usefulness of the HCVcc system. More importantly, virus recovered from HCVcc inoculated animals (ex vivo HCVcc; chHCVcc and muHCVcc for virus recovered from chimps and mice, respectively) was infectious in cell culture establishing the first robust ex vivo culture system. Virus recovered from infected animals displayed both altered biophysical properties and increased specific infectivity (ratio of infectious units to RNA copies) indicating that ex vivo HCVcc is of great use in elucidating the role of host factors in modulating HCV infection.
Using HCVpp, much has been learned about the mechanism of HCV cell entry. Evaluations of entry using the HCVcc system yield comparable results with regard to cell entry properties such as CD81 dependence, restriction to human hepatoma cell lines and neutralization by anti-E2 antibodies. Even with HCVcc available, HCVpp still offer certain advantages, most notably (1) the ability to investigate HCV glycoprotein dependent entry in cells non-permissive to HCV replication and (2) the availability of stringent controls in the form of pseudoparticles bearing glycoproteins from viruses other than HCV, such as VSV or MLV, and pseudoparticles devoid of glycoproteins (no envelope). Nonetheless, future studies are needed to elucidate possible mechanistic differences between the cell entry properties of HCVpp, HCVcc and ex vivo HCVcc.
HCVpp infectivity requires both E1 and E2 with their intact transmembrane domains (Bartosch, B. J., et al. 2003 J Exp Med 197:633-642; Hsu, M., et al. 2003 Proc. Natl. Acad. Sci. USA 100:7271-76). The structure of the infectious unit in vivo may be more complex through the above mentioned association between the virus and host serum factors including different lipoprotein species (VLDL, LDL, HDL) and immunoglobulins (Kono, Y., J et al. 2003 Med Virol 70:42-8; Monazahian, M., et al. 2000 Journal of Medical Virology 57:223-9; Thomssen, R., et al. 1992 Med. Microbiol. Immunol. 181:293-300; Thomssen, R., et al. 1993 Med. Microbiol. Immunol. 182:329-334). Such associations may explain the heterogeneous buoyant density observed for both plasma-derived HCV (1.03-1.2 g/ml) (Bradley, D., J. et al. 1991 Med. Virol. 34:206-208; Hijikata, M., J. et al. 1993 Virol. 67:1953-1958; Thomssen, R., et al. 1993 Med. Microbiol. Immunol. 182:329-334) and HCVcc (1.04-1.18 g/ml) (Lindenbach, B. D., et al. 2005 Science 309:623-6; Wakita, T., et al. 2005 Nat Med 11:791-6; Zhong, J., et al. 2005 Proc Natl Acad Sci USA 102:9294-9). The highest infectivity seems to be associated with fractions of low to medium density (1.11 g/ml and below) (Bradley, D., J. et al. 1991 Med. Virol. 34:206-208; Hijikata, M., J. et al. 1993 Virol. 67:1953-1958; Lindenbach, B. D., et al. 2005 Science 309:623-6) indicating that an interaction with plasma lipids may enhance virion infectivity.
Low pH induces conformational changes in E2 and the dissociation of E1E2 complexes indicating the involvement of a pH-triggered step in the entry process (Flint, M., et al. 1999 J. Virol 73:6782-6790; Op De Beeck, A., et al. 2004 J Virol 78:2994-3002). In keeping with this, HCVpp entry has been shown to be sensitive to endosomal acidification inhibitors such as ammonium chloride, bafilomycin and concanamycin, as is HCVcc entry, as shown herein. This pH dependence indicates that virus interaction with putative cell surface receptors is followed by endocytotic uptake of the particle rather than fusion at the plasma membrane and that endosomal low pH is required, in some embodiments, to initiate virus-cell membrane fusion. An endosomal route of entry has also been described for the related flaviviruses (Gollins, S. W., et al. 1985 J. Gen. Virol. 66:1969-1982; Gollins, S. W., et al 1986 J. Gen. Virol. 67:157-166) as well as more evolutionarily distant alphaviruses (Helenius, A., et al. 1980 J Cell Biol 84:404-20).
Currently, the minimal host cell factor requirement for HCV cell entry (i.e., the sequence of events beginning with attachment to the host cell and ending with cytoplasmic delivery of the nucleocapsid) is not known. Numerous molecules have been proposed to function as HCV (co-)receptors (i.e., cell surface molecules required for entry that bind virus). However, none of these have had a precise function in the entry process conclusively defined, nor has the temporal sequence of interactions required for entry been determined. Beyond (co-) receptors, additional molecules that perform other functions in the entry process (e.g., endosomal proteases) may be required. Finally, there may also be molecules that are not essential but rather facilitate HCV entry (facilitating factors).
There appears to be a requirement for CD81 for HCV cell entry. CD81, a member of the tetraspanin superfamily with four transmembrane domains and short cytosolic N- and C-terminal tails, was initially identified as a candidate HCV receptor based on its ability to bind sE2124 The HCV-CD81 interaction is thought to take place between the CD81 large-extracellular loop (LEL) between transmembrane domains 3 and 4 (Drummer et al., 2005, Biochem Biophys Res Commun 328:251-7; Drummer et al., 2002, J Virol 76:11143-7; Higginbottom et al., 2000, J Virol 74:3642-9) and a conformational epitope on E252. Several pieces of evidence strongly support CD81's role as an essential (co-)receptor for HCV: (1) The human hepatoma cell line HepG2 does not express CD81 and cannot be infected with HCVpp or HCVcc, but becomes infectable with both upon transduction with CD81 (Bartosch et al., 2003, J Biol Chem 278:41624-30; Lindenbach et al., 2005, Science 309:623-6; Zhang et al., 2004, J Virol 78:1448-55). This CD81 requirement in HepG2 cells is conserved across HCVpp bearing E1 and E2 from all known genotypes (Lavillette et al., 2005, Hepatology 41:265-74; McKeating et al., 2004, J Virol 78:8496-505). (2) Knockdown of CD81 expression using siRNA abrogates susceptibility to HCVpp166. (3) Antibodies against CD81, as well as soluble forms of the large extracellular loop of CD81, block HCVpp and HCVcc infection in a dose-dependent manner (Bartosch, B., et al. 2003 J Exp Med 197:633-642; Hsu, M., et al. 2003 Proc. Natl. Acad. Sci. USA 100:7271-76; Lindenbach, B. D., et al. 2005 Science 309:623-6; Wakita, T., et al. 2005 Nat Med 11:791-6; Zhong, J., et al. 2005 Proc Natl Acad Sci USA 102:9294-9). However, other factors besides CD81 must be required for entry since CD81 expression alone is insufficient to allow HCVpp entry (Bartosch, B., et al. 2003 J Exp Med 197:633-642; Hsu, M., et al. 2003 Proc. Natl. Acad. Sci. USA 100:7271-76; Zhang, J., et al. 2004 J Virol 78:1448-55) and the expression of CD81 in all human cell types except erythrocytes and platelets (Levy, S., et al. 1998 Annu. Rev. Immunol. 16:89-109) does not explain HCV's apparent liver tropism. The precise role of CD81 in the entry process is unclear; some evidence suggests it may function as a co-receptor, interacting with the virus only after binding of the virus to another receptor molecule has occurred (Cormier, E. G., et al. 2004 Proc Natl Acad Sci USA 101:7270-4).
Like CD81, scavenger receptor class B member I (SR-BI) was first proposed as an HCV entry factor because of its ability to bind sE2136. SR-BI is expressed at high levels in the liver and steroidogenic tissues with lower levels detectable in placenta, small intestine, monocytes/macrophages and other tissues. It mediates selective uptake of cholesterol esters from HDL into the cellular membrane (Acton, S., et al. 1996 Science 271:518-20; Rodrigueza, W. V., et al. 1999 J. Biol Chem 274:20344-50) and possibly also endocytosis of entire HDL particles (Silver, D. L., et al. 2001 J Biol Chem 276:25287-93). The role of SR-BI in HCV cell entry is less clear than that of CD81. No SR-BI negative cell line that becomes permissive to HCV infection when transfected with SR-BI has been reported. Antibodies and siRNA directed against SR-BI inhibit HCVpp infection (Bartosch et al., 2003, J Biol Chem 278:41624-30; Lavillette et al., 2005, Hepatology 41:265-74), but both effects are less striking than those obtained for CD81 and vary considerably between HCV genotypes (Lavillette et al., 2005, Hepatology 41:265-74) (and unpublished data). Recently, HCVpp infectivity was found to be enhanced significantly in the presence of HDL (Bartosch et al., 2005, J Virol 79:8217-29; Meunier et al., 2005, Proc Natl Acad Sci USA 102:4560-5; Voisset et al., 2005, J Biol Chem 280:7793-9). The enhancement depends on functional SR-BI on the target cell since both SR-BI siRNA and BLT-4, a drug that inhibits flux of cholesteryl esters from SR-BI bound HDL into the target cell membrane_(Nieland, T. J., et al. 2002 Proc Natl Acad Sci USA 99:15422-7), completely abrogate the enhancing effect of HDL. These treatments have no (BLT-4) or variable (siRNA) effects on infectivity in the absence of HDL (Bartosch, B., et al. 2005 J Virol 79:8217-29; Voisset, C., et al. 2005 J Biol Chem 280:7793-9). Finally, it was found that oxidized LDL, an LDL-derived product of atherosclerotic processes and a known SR-BI ligand, dramatically inhibits HCVpp and HCVcc infectivity (Hahn, T., et al. 2006 Hepatology In press). Based on these findings, it would appear that, in addition to CD81, SR-BI also has an important role in HCV entry. However, co-expression of CD81 and SR-BI is not sufficient to confer susceptibility to HCVpp (Bartosch, B., A., et al. J Biol Chem 278:41624-30; Hsu, M., J. et al. 2003. Proc. Natl. Acad. Sci. USA 100:7271-76), suggesting that additional factors are required.
The C-type lectins dendritic cell- and liver-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN and L-SIGN) binds E2 (Gardner et al., 2003, Proc Natl Acad Sci USA 100:4498-4503; Lozach et al., 2003, J Biol Chem 278:20358-66; Pohlmann et al., 2003, J Virol 77:4070-4080), HCVpp (Cormier et al., 2004, Proc Natl Acad Sci USA 101:14067-72; Lozach et al., 2004, J Biol Chem 279:32035-45) and several other viruses (Alvarez et al., 2002, J Virol 76:6841; Geijtenbeek et al., 2000, Cell 100:587-97; Halary et al., 2002, Immunity 17:653-64; Tassaneetrithep et al., 2003, J Exp Med 197:823-9). The interaction between HCVpp and a cell expressing DC- or L-SIGN does not result in infection; however, bound HCVpp can be transmitted to permissive cells in co-culture (Cormier et al., 2004, Proc Natl Acad Sci USA 101:14067-7; Lozach et al., 2004, J Biol Chem 279:32035-45), as is the case for HIV (Geijtenbeek et al., 2000, Cell 100:587-97). As L-SIGN and DC-SIGN are expressed on liver sinusoidal endothelial cells and DCs, respectively, a model where they capture and transmit HCV particles to susceptible hepatocytes is feasible but unproven. The association of HCV with lipoproteins (Monazahian et al., 2000, Med Microbiol Immunol (Berl) 188:177-84; Thomssen et al., 1992, Med. Microbiol. Immunol. 181:293-30) has led to the hypothesis that the low density lipoprotein receptor (LDL-R) may be involved in HCV entry. At least in the presence of plasma, LDL-R appears to mediate cell attachment and possibly cellular uptake of plasma derived HCV RNA (Agnello et al., 1999, Proc. Natl. Acad. Sci. USA 96:12766-12771; Monazahian et al., 1999, Journal of Medical Virology 57:223-9; Wunschmann et al., 2000, J Virol 74:10055-62). Whether this interaction results in productive infection, however, is uncertain, as HCVpp do not seem to require LDL-R for cell entry (Bartosch et al., 2003, J Exp Med 197:633-642; Hsu et al., 2003, Proc. Natl. Acad. Sci. USA 100:7271-76). Moreover, heparan sulfates (HS) (Barth, H., et al. 2003 J Biol Chem 278:41003-12) and asialoglycoprotein receptor (ASGP-R) (Saunier, B., et al. 2003 J Virol 77:546-59) have been suggested as HCV entry factors, but their roles have not been rigorously validated in an infection assay.
When HCVpp became available it was quickly noted that only a select group of cell lines, all of which were derived from human liver, could be infected (Bartosch et al., 2003, J Exp Med 197:633-642; Hsu et al., 2003, Proc. Natl. Acad. Sci. USA 100:7271-76; Zhang et al., 2004, Virol 78:1448-55). What precisely defines this narrow tropism is as yet unclear. So far, no set of molecules sufficient to permit HCVpp entry into a target cell has been defined. Indeed, several cell lines express CD81, SR-BI and LDL-R at levels comparable to permissive cells and still cannot be infected (Hsu, M., et al. 2003 Proc. Natl. Acad. Sci. USA 100:7271-76). Thus one or more additional factor(s) essential for HCV entry are still missing.
In 1998, Furuse and colleagues identified CLDN1 and Claudin 2 (CLDN2) as integral membrane proteins present in the tight junctions of mouse hepatocytes that were able to reconstitute de novo tight junction (TJ) strands when expressed in mouse fibroblasts (Furuse, M., et al. 1998 J Cell Biol 141:1539-50; Furuse, M., et al. 1998 J Cell Biol 143:391-401). Subsequently, several homologous gene products were identified bringing the claudin gene family up to 24 members (Van Itallie, C. M., and J. M. Anderson. 2005. Claudins and Epithelial Paracellular Transport. Annu. Rev Physiol. for review). Claudins are small (20-27 kD) molecules with a short cytoplasmic N- and C-terminal tails. Four membrane-spanning helices are separated by a larger (˜53aa) first and a smaller (˜24aa) second extracellular loop (EL1 and EL2, respectively) and a very short intracellular loop (FIG. 3).
Claudins are thought to be the major structural component of the TJ in epithelia where claudin family members and other TJ associated membrane proteins such as occludin, the junction-adhesion-molecule (JAM) and the coxsackie-adenovirus-receptor (CAR) associate laterally to form the TJ strand (Furuse, M. et al. 1999 J Cell Biol 147:891-903; Gonzalez-Mariscal, L. et al. 2003 Prog Biophys Mol Biol 81:1-44). TJ strands in the membrane of neighboring cells then interact across the intercellular space to form the actual TJ. The extracellular loops of the claudins are thought to be central to these intercellular contacts that narrow and largely obliterate the intercellular space, thus forming the barrier between the apical and the basolateral side of the epithelium (Furuse, M., et al. 1999 J Cell Biol 147:891-903). Despite their name, TJs do not completely seal off the paracellular pathway but allow selective flux of solutes based on size and charge. Paracellular pathway selectivity seems to be determined largely by the extracellular domains of the claudins present in a given tight junction (Colegio, O. R., et al. 2003 Am J Physiol Cell Physiol 284:C1346-54; Furuse, M., et al. 2001. J Cell Biol 153:263-72). Thus, the modular claudin composition of the tight junction may determine both the transepithelial electrical resistance and the paracellular pathway selectivity for certain solutes in epithelial tissues. Finally, through their intracellular C-terminus, claudins interact with PDZ-domain containing adaptor proteins, such as the TJ associated proteins zonula occludens (ZO)-1, -2 and -384.
CLDN1 is expressed in a number of epithelia, with the highest levels detected in the liver followed by kidney, skin and other tissues (Furuse, M., et al. 1998 J Cell Biol 141:1539-50; Su, A. I., et al. 2002 Proc Natl Acad Sci USA 99:4465-70). Claudin-1 knockout mice die in the neonatal period due to water loss through the skin (Furuse, M., et al. 2002 J Cell Biol 156:1099-111). However, loss of CLDN1 is tolerated in humans; individuals homozygous for a two nucleotide deletion in the Claudin-1 gene resulting in frame shift and a premature stop codon have been found in two inbred families of Moroccan descent (Hadj-Rabia, S., et al. 2004 Gastroenterology 127:1386-90). Affected patients exhibit scaling skin (ichthyosis) and liver disease due to neonatal sclerosing cholangitis. Moreover, several other members of the claudin family have been implicated in genetic and infectious diseases: Claudin-14 mutations cause recessive non-syndromic deafness (Wilcox, E. R., et al. 2001 Cell 104:165-72) and this phenotype was replicated in Claudin-14 knockout mice_(Ben-Yosef, T., et all. 2003 Hum Mol Genet. 12:2049-61). Defects in Claudin-16 (Paracellin) result in renal magnesium loss in humans (Simon, D. B., et al. 1999 Science 285:103-6). The C-terminus of the Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, binds specifically to Claudin-3 and -4 causing disruption of intestinal TJs while the N-terminus forms pores in the plasma membrane leading to further disruption of epithelial integrity (Fujita, K., et al. 2000 FEBS Lett 476:258-61; Hanna, P. C., et al. 1992 Infect Immun 60:2110-4; Sonoda, N., et al. 1999 J Cell Biol 147:195-204). As mentioned above and described in section C, we have evidence indicating that CLDN1 is a required entry factor for HCV. Interestingly, several other TJ molecules have been implicated in viral infection; CAR functions as a receptor for coxsackie- and adenoviruses (Bergelson, J. M., et al. 1997 Science 275:1320-3); JAM is an essential receptor for reoviruses (Barton, E. S., et al. 2001 Cell 104:441-51); and CLDN7 has been implicated in HIV entry into CD4 negative cells (Zheng, J., et al. 2005 Retrovirology 2:79). A recent elegant study expanded on this theme of TJ components in viral entry by showing that group B coxsackie virus initially engages a receptor, DAF, on the luminal surface of intestinal cells. DAF binding triggers signaling events that result in the migration of the virus-DAF complex to the TJ where an interaction with a co-receptor, CAR, occurs that then results in caveolin dependent uptake of the viral particle (Coyne, C. B., et al. 2006 Cell 124:119-31).